Mycobacterium avium subsp. paratuberculosis exploits miRNA expression to modulate lipid metabolism and macrophage polarisation pathways during infection

Pathogenic mycobacteria including Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease, manipulate host macrophages to persist and cause disease. In mycobacterial infection, highly plastic macrophages, shift between inflammatory M1 and permissive M2 phenotypes which alter the disease outcome and allow bacteria to survive intracellularly. Here we examine the impact of MAP infection on polarised macrophages and how increased lipid availability alters macrophage phenotype and bacterial persistence. Further, we assess if host microRNA (miRNA) are sensitive to macrophage polarisation state and how MAP can drive their expression to overcome innate responses. Using in vitro MAP infection, we find that increasing lipid availability through supplementing culture media with exogenous lipid increases cellular nitric oxide production. Lipid-associated miRs -19a, -129, -24, and -24-3p are differentially expressed following macrophage polarisation and lipid supplementation and are further regulated during MAP infection. Collectively, our results highlight the importance of host lipid metabolism in MAP infection and demonstrate control of miRNA expression by MAP to favour intracellular persistence.

The interaction between macrophages and virulent mycobacteria is a major determining factor in the outcome of infection. Macrophages form one of the first lines of defence against invading pathogens and are often hijacked by pathogenic mycobacteria to provide a survival niche, protected from the host immune system 1 . Macrophages may be categorised into subsets, including either classically activated proinflammatory M1, or alternatively activated anti-inflammatory M2 phenotypes. Macrophage polarisation is an adaptable process and is determined by the type of immune stimuli and pathogen signal 2 . During mycobacterial infection there is, however, a high degree of plasticity in macrophages, often indicative of disease state and stage of infection 3,4 .
Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic mycobacterium which causes the enteric granulomatous disease, Johne's disease. Following uptake via the faecal oral route, MAP is initially able to cross the intestinal epithelial barrier via specialised microfold or M cells within Peyer's patches where it is then phagocytosed by macrophages 5 . A hallmark of pathogenic mycobacteria including MAP is the ability to manipulate a range of macrophage functions, including preventing phagosome maturation, lysosomal degradation, and preventing macrophage apoptosis [6][7][8] . This blockade of innate macrophage defences allow MAP to persist within host macrophages, leading to the formation of granulomatous lesions and long-term chronic infections. Studies on the polarisation of host macrophages during MAP infection have identified mixed populations of both M1 and M2 macrophages rather than a distinct classical or alternative dominance 9 . Further, macrophage polarisation state is largely related to disease state, with clinically infected animals showing lower numbers of microbicidal M1 macrophages, while subclinically infected animals display similar of numbers of M1 and M2 "regulatory" macrophages 10 .

Results
Murine macrophage polarisation. Macrophage differentiation was assessed by gene expression analysing common M1 and M2 markers (Nos2 and Arg-1 respectively), as well as cellular nitric oxide (NO) secretion, while MAP infection was confirmed microscopically. As expected, M1 but not M2 polarised macrophages produced high levels of NO (Fig. 1a). To characterise the effect on macrophage phenotype following supplantation with exogenous lipid, unpolarised macrophages were supplemented with lipid and cellular NO production was measured and compared to LPS (positive control). At 6 h, lipid supplementation resulted in an increase in cellular NO production in unstimulated macrophages on par with positive control levels. This increase in cellular NO was also observed at 24 h, indicating that macrophages provided with exogenous lipid are shifted towards an inflammatory M1 phenotype (Fig. 1b).
To further characterise the activation state of polarised macrophages supplemented with lipid and/or infected with MAP, the expression of M1 and M2 marker genes Nos2 and Arg-1 was measured. In line with observed NO production, M1 polarised macrophages displayed increased expression of the M1 marker Nos2 and low expression of M2 marker Arg-1, while the combination of lipid and MAP infection did not alter the phenotype or activation state of these cells (Fig. 1c,d). In M1 polarised macrophages, lipid and MAP reinforced the M1 phenotype, however these factors had the opposing effect in M2 differentiated cells, shifting the macrophages away from the M2 phenotype.
Macrophages supplemented with lipid were imaged at 6 and 24 h post infection (hpi) to assess phenotype and foam cell formation. At both 6 and 24 hpi, visible foam cells were observed compared to control macrophages. When lipid supplemented macrophages were infected with MAP, foam cells were still observed at 6 hpi, however bacterial clearance was reduced compared to non-supplemented MAP infected macrophages (Fig. 2). Overall, this suggests that MAP infection and lipid supplementation drive an M1 inflammatory macrophage phenotype in early infection, even in previously M2 polarised macrophages, and the formation of foam cells during MAP infection is beneficial for infection.  ing key lipid biosynthesis, transport, and cholesterol metabolism genes abca1, abcg1, apoa1, apoe, apob, and ldlr (Table 1) was performed. miRNA-mRNA pairs that appeared in at least two software predictions databases and were either experimentally observed or moderate-high predicted targeting were chosen to investigate their role in driving macrophage phenotypes and their interaction with MAP (Table 2).
Murine miRNA gene expression. The effect of MAP infection and macrophage polarisation on miRNA expression was initially investigated using the murine macrophage cell line, RAW 264.7 . Ten miRNAs bioinformatically selected as having a role in lipid metabolism pathways and/or macrophage polarisation and activity were profiled at 24 hpi (Figs. 3, 4). miR-129 expression was significantly decreased in MAP infected cells, however remained upregulated in all M1 polarised cells in comparison (Fig. 3a), potentially indicating the involvement of miR-129 in the response to MAP, that is further sensitive to M1 polarisation state and lipid supplementation. Similarly, miR-24-3p was significantly upregulated in all non-infected macrophages compared to MAP infected cells, regardless of polarisation phenotype or lipid supplementation (Fig. 3b). miR-24-3p was downregulated in all MAP infected macrophages, indicating a potentially MAP-driven response to suppress expression. Likewise, miR-144 was upregulated in all non-infected cells, except for M2 + lipid treatment, in comparison to MAP only cells (Fig. 3c). The addition of MAP to macrophages downregulated miR-144 in both M1 and M2 macrophages including those with lipid supplementation.
Expression of miR-24 was upregulated in M1 polarised uninfected macrophages and in all MAP infected cultures compared to control uninfected cells, with the exception of the control MAP infected cultures without lipid supplementation (Fig. 3d). This suggests that MAP, and MAP with access to excess lipid, may be driving expression of miR-24. However, there were no significant differences in miR-24 expression between the treatment groups (polarised, MAP-infected and/or lipid supplemented cultures). miR-19a showed similar expression patterns and was upregulated in M1 macrophages and lipid supplemented macrophages compared to both  www.nature.com/scientificreports/ control uninfected macrophages, MAP infected, and M2 polarised infected and lipid supplemented macrophages (Fig. 3e). As lipid supplemented cells displayed an M1 phenotype with increased NO production ( Fig. 1), miR-19a may be a regulator of lipid metabolism in MAP infection, with bacteria suppressing this response in infected and M2 macrophages. While expression of miR-148a was upregulated in M1 polarised MAP infected cells, with or without lipid supplementation compared to control uninfected cells, there were no significant differences in expression between treatment groups (Fig. 3f), implying that miR-148a is associated with M1 inflammatory antimycobacterial macrophage responses. A similar M1 polarisation response was observed for expression of miR-19b, with upregulation in M1 and lipid supplemented cells compared to controls, however there were no significant differences found between the various treatment groups (Fig. 4a). miR-455 was upregulated in lipid supplemented macrophages with the exception of the M2 + lipid cultures, as well as in M1 polarised cells that were supplemented with either lipid or MAP, compared to the control uninfected cells (Fig. 4b). However, there was no significant regulation of this miRNA between polarised cells and those supplemented with MAP or lipid. Although miR-425 was upregulated in M1 infected macrophages compared to control uninfected cells, the expression between the treatment groups was not significantly different (Fig. 4c). Expression of miR-758 was not significantly regulated in macrophages following any treatment or supplementation (Fig. 4d).
Many of the miRNA were either strongly upregulated or downregulated in infected or lipid supplemented macrophages, as summarised in Table 2. Further analysis of their involvement in immune responses to infection may provide further understanding of the involvement of MAP in host lipid metabolism. Hence, miRs -19a, -24, -24-3p, and -129 were chosen as candidates for further investigation in bovine cells due to their modulation of expression by MAP.
Bovine miRNA gene expression. Expression levels of four miRNA chosen for further investigation from murine studies was profiled in the BoMac cell line following MAP infection (Table 3). As the addition of MAP and/or lipid supplementation resulted in a primarily M1 inflammatory phenotype, regardless of previous polarisation status, we chose to assess miRNA expression only in MAP infected or control bovine macrophages.
At 4 hpi, miRs -19a, -24-3p, and -129 were all significantly upregulated in infected cells compared to uninfected controls. At 20 hpi, miRs -19a and 24-3p remained increased, while expression of miR-129 was decreased. Despite a trend towards a decrease in transcript abundance of miR-24 at 4 hpi, there was no significant regulation at either timepoint (Fig. 5a).
To further confirm the relevance of expression profiles of these miRNA in primary cells, bovine macrophages were infected with MAP and miRs -24, -24-3p, -129a, and -19a quantified (Fig. 5b). In contrast to miRNA expression in MAP infected BoMac cells, primary bovine macrophages infected with MAP displayed a miRNA Table 2. Change in miRNA expression in MAP infected murine macrophages. Red boxes indicate upregulated miRNA and green boxes represent downregulated miRNA compared to control uninfected cells. While boxes indicate no significant differential expression. Log2 fold changes ± 1.5 compared to control uninfected cells was considered differentially regulated.  www.nature.com/scientificreports/ expression profile which was similar to the regulation in MAP infected murine macrophages. miR-19a and miR-24-3p were increased at both timepoints in infected primary bovine cells, whereas miR-24 and miR-129 displayed a switch from upregulated to downregulated from 4 to 20 hpi. Table 3 summarises miRNA expression in MAP infected BoMac cells and primary bovine macrophages, showing the modulation of transcription during the early stages of infection.
In MAP infected BoMac cells, similar expression patterns to murine RAW cell were observed for miR-19a and miR-129 at later timepoints (20-24 hpi), while miR-24 showed no regulation in either direction. Interestingly, miR-24-3p displayed opposing regulation in MAP infected in mouse and bovine cells, potentially due to variations in species-specific target binding and differing functional arms of pre-miRNA.

Discussion
Macrophages are central to the phagocytosis and clearance of MAP by immune cells. However, they are also commandeered into providing an intracellular niche for the bacteria to persist and sustain infection 1 . Specific macrophage phenotypes direct the responses to invading pathogens. Broadly, the signals macrophages receive from their cellular milieu drive this polarisation. M1 polarised macrophages are pro-inflammatory and microbicidal, producing large amounts of TNF-α and NO. M2 macrophages are often anti-inflammatory and fail to kill mycobacteria 25,26 . It is therefore clear that polarisation of macrophages influences the outcome of infection at a cellular level and is likely to impact pathogenesis and disease outcome at the level of the whole animal 2 . In addition to host immune effectors, MAP itself can divert macrophage phenotype and therefore function to its benefit. Through modulation of miRNA transcript abundance, pathogenic mycobacteria are able to alter downstream www.nature.com/scientificreports/ target mRNA expression to moderate host immune responses 27 . miRNA provide another level of regulation through which mycobacteria can co-opt host signalling pathways to prevent clearance and establish infection [28][29][30] .
In this study, we aimed to investigate the effect of lipid supplementation on macrophage polarisation and miRNA expression to reveal MAP driven responses to regulate host lipid metabolism. The role of lipids in macrophage polarisation is of particular interest, as MAP and mycobacteria actively utilise host lipid pathways to aid persistence and block host defence mechanisms [14][15][16][17]31 . Lipogenesis and fatty acid synthesis are key processes in inflammatory immune responses, driving macrophages towards an M1 phenotype and activating microbicidal inflammasomes [32][33][34] . Conversely, lipolysis is believed to be a driver of M2 macrophage functions, perpetuating an anti-inflammatory response 35 . The interaction between these host processes and bacterial survival strategies may alter immune responses to MAP.
We found that lipid supplementation skewed macrophage phenotypes towards an M1 state. As lipid supplementation resulted in foam cell formation and reduced bacterial clearance in M1 polarised and supplemented macrophages, we concluded that inflammatory macrophages and utilisation of host lipids were key pathways regulated by MAP following infection. Further investigation of post-transcriptional regulation of these pathways provided four miRNA which appear to be responsive to infection and associated with lipid related functions.  www.nature.com/scientificreports/ Previous studies have associated miR-129 with macrophage polarisation and control of mycobacteria through targeting of SOCS2 and the Sp2 transcription factor [36][37][38] . Decreased miR-129 transcripts at 20-24 hpi in MAP infected macrophages may promote eventual M2 polarisation through increased SOCS2 and Sp2 target expression, responsible for IL-10 induction. During early infection (4 hpi), miR-129 was upregulated, likely maintaining the M1 phenotype, before switching to a pro-survival M2 phenotype in the later stages of early pathogenesis (20-24 hpi). Further, miR-129 has been shown to regulate autophagy through ATG7 and HMGB1, potentially facilitating disruption of the phagolysosomal pathway by MAP 39,40 . Supplementation of macrophages with exogenous lipid rescued the M1 polarisation phenotype in MAP infected cells, indicating a lipid dependant function of miR-129. A potential target of miR-129, transcription factor Sp2, is known to regulate cholesterol and lipid biosynthetic pathways 41 , and may explain the increased bacterial clearance observed in M1 polarised lipid rich macrophages.
miR-24-3p elicited a similar response to lipid supplementation and MAP infection in murine macrophages but not bovine macrophages. In murine macrophages infected with MAP, miR-24-3p expression was reduced compared to non-infected cells, regardless of polarisation state or lipid supplementation. While this infection-driven reduction in miR-24-3p transcription was not apparent in MAP infected bovine macrophages, there was a trend towards reduced expression from early to later timepoints, however, may represent unrelated temporal changes in expression rather than MAP-dependant changes. miR-24 is highly conserved between species, and while both miR-24 and miR-24-3p arise from the same pre-miRNA molecule, they may possess different functional capabilities across species. Expression of miR-24 in bovine cells was similar to that of miR-24-3p, suggesting that they may share functions and bioactivity rather than being independently regulated as observed in murine infections.
As miR-24-3p contributes to the attenuation of phagocytosis and promotes alternative or M2 macrophage activation [42][43][44] , increased miR-24-3p following MAP infection may promote an M2 macrophage state, suppressing the MAP-associated inflammatory phenotype. Further suggested roles for miR-24-3p include interference with antigen presentation in myeloid cells and the regulation of apoptotic pathways, further supporting the apparent mechanism of miR-24-3p in suppressing the infection-associated M1 inflammatory phenotype in host macrophages following MAP infection [45][46][47] . Further, miR-24-3p has been shown to suppress heme oxygenase (hmox), which has recently been implicated in macrophage migration in mycobacterial infection, suggesting that altered miR-24-3p expression may be impacting macrophage activation and activity 48,49 .
Another miRNA of interest from our work is miR-19a, an apparent M1 inflammatory marker. In murine macrophages, miR-19a was strongly upregulated in M1 and lipid supplemented cells while the magnitude of this increase was dampened following infection with MAP. An increase in MAP infected macrophages may contribute to the observed foam cell formation and reduction in cellular lipid efflux. miR-19a acts on several lipid metabolism pathways to regulate lipid efflux and mediate inflammation, while reduced expression in MAP infected macrophages suggests this infection-responsive miRNA may be modulated by MAP to promote survival. Through a reduction in its targets 5-lipoxygenase, HBP-1, and PPAR-α, miR-19a is able to reduce lipid efflux and catabolism, promoting foam cell formation and providing potential metabolic fuel to intracellular MAP 36,[50][51][52][53] .
The interaction between MAP and macrophages is paramount to host control and disease progression, and the complex relationship between mycobacteria and host lipids further complicate immune responses. Infection with MAP led to an M1 inflammatory response, suggesting that during early pathogenesis MAP may utilise lipid pathways to support the M1 macrophage state and in turn, their persistence within cells. Changes in miRNA expression throughout the progression of early infection further suggests that MAP is able to manage host miRNA responses to change the cellular microenvironment as infection progresses.
The ability of MAP and virulent mycobacteria to modulate gene expression and host lipid metabolism pathways for their survival is a multifaceted process; however, we have shown that there is a further level of regulation by non-coding miRNA. This provides MAP with another means to control host gene expression and alter effective immune function. As summarised in Fig. 6, MAP infection is able to alter host miRNA expression to affect downstream targets; however, the pleiotropic nature of miRNA and their multitude of targets makes interpretation of effector pathways difficult. This further highlights the need for context-dependant functional studies as miRs may exhibit differing regulation under different biological conditions. This study utilised several in vitro models to uncover MAP-driven miRNA dysregulation. While murine RAW264.7 macrophages and primary bovine macrophages displayed similar expression profiles, there was a discrepancy between BoMac cells and primary bovine macrophages. Although disparities in the magnitude of expression and directionality between murine and bovine cells can be partially explained by species-specific miRNA roles; the differential expression between BoMac and primary macrophages suggests activation of alternate phagocytic pathways. BoMac cells lack cell surface CD14 receptors, several integrin receptors and have lower phagocytic capacity for MAP as well as a reduced ability to allow its intracellular replication 54,55 . The combination of these factors may explain the differences in induction or inhibition of miRNA, with the interaction of MAP and BoMac cells activating non-CD14/TLR dependant phagocytosis and engulfment, which leads to alternate signalling pathways and intracellular conditions 55,56 . The use of these multiple in vitro models of MAP infection provides further insight into the cellular cues which drive miRNA expression during infection. These results provide an interesting starting point for the investigation of key pathogen recognition and signalling molecules involved in MAP-dependant miRNA expression.
Analysis of infection-induced gene expression has further contributed to the growing knowledge of miRNA control of host immune responses, and their alteration following mycobacterial infection. While we selected only a few miRNAs to profile, further studies to uncover lipid-associated miRs may provide key molecules and pathways altered during infection and provide potential therapeutics for the treatment of mycobacterial infection. To conclude, we have shown that miRs -129, -24-3p, and -19a are responsive to macrophage polarisation and that MAP is able to alter their expression to exploit lipid metabolism and macrophage polarisation pathways.
A standard curve was included for each miRNA on each plate using a fivefold dilution of neat cDNA. Amplicon specificity was confirmed with a dissociation curve and the standard curve analysed to ensure efficiency between 90-110%, slope between 3.1-3.6, and R 2 > 0.98. Data was normalised to either β-actin or U6 and analysed using the 2 -ΔΔ Ct method. www.nature.com/scientificreports/ Griess test. A Griess assay was performed to assess nitric oxide secretion from polarised cells. The Griess test measures nitrite within cell culture supernatant to evaluate the NO production. Briefly, 1 × Griess Reagent (modified) (Sigma-Aldrich, USA) was prepared by adding 250 mL of ultrapure Milli-Q water to the bottle and mixed by inverting. A standard curve was generated for each plate using a serial dilution of 97% sodium nitrite (Sigma-Aldrich, USA) and serum-free culture media. Culture supernatant (50 µL) from each sample (in duplicate) mixed with 50 µL of 1 × Griess Reagent. Plates were covered from light and gently mixed for 2 min using a plate shaker and incubated in the dark for 50 min. Absorbance at 540 nm was measured using a plate reader.
Statistical analysis. Statistical analysis was performed in GraphPad Prism (v.9.0.0) using default parameters. For qPCR analysis, raw Cq values were normalised to housekeepers U6 pseudogene (miRNA analysis) or β-actin (mRNA analysis) and analysed using the 2 −ΔΔ Ct method 59 . Genes were considered differentially expressed if there was a statistically significant difference in the fold change compared to control. P values were calculated using a one-way ANOVA followed by Tukey's post-hoc analysis to determine differences between pairwise comparisons, with significance at the 5% level. P values are represented by asterisk's: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Data availability
Analysed data are presented within the current manuscript. Individual raw data is available from the corresponding author upon reasonable request.